Expression cloning methods in filamentous fungi

ABSTRACT

Methods for screening a polynucleotide library for a polypeptide with a property of interest in a filamentous fungal host cell, in a manner which allows quick and easy subsequent characterization of the polypeptide, using an expression cloning vector comprising at least a polynucleotide encoding a selectable marker in which the translation initiation start site of the marker-encoding sequence comprises a crippled consensus Kozak sequence, a fungal replication initiation sequence, and a promoter with a cloning-site into which the library is cloned, and a transcription terminator.

BACKGROUND OF THE INVENTION

Several methods for the construction of libraries of polynucleotidesequences of interest in yeast have been disclosed in which thelibraries are screened in yeast prior to transformation of anindustrially relevant filamentous fungal host cell with a selectedpolynucleotide.

Often however, a polynucleotide sequence identified by screening inyeast or bacteria cannot be expressed or is expressed at low levels whentransformed into production relevant filamentous fungal cells. This maybe due any number of reasons, including differences in codon usage,regulation of mRNA levels, translocation apparatus, post-translationalmodification machinery (e.g., cysteine bridges, glycosylation andacylation patterns), etc.

A. Aleksenko and A. J. Clutterbuck (1997. Fungal Genetics and Biology21: 373-387) disclose the use of autonomous replicative vectors, orautonomously replicating sequences (ARS), for gene cloning andexpression studies. AMA1 (autonomous maintenance in Aspergillus) is oneof the plasmid replicator elements discussed. It consists of twoinverted copies of a genomic repeat designated MATE1 (mobile Aspergillustransformation enhancer) separated by a 0.3 kb central spacer. AMA1promotes plasmid replication without rearrangement, multimerization orchromosomal integration. AMA1-based plasmids provide two advantages ingene cloning in filamentous fungi. The first is a high frequency oftransformation which both increases the potential library size and caneliminate the need for library amplification in an intermediate host,e.g., E. coli, so that a recipient Aspergillus strain can be transformeddirectly with a ligation mixture. Secondly, by providing a stable andstandard environment for gene expression, the properties of thetransformants will be uniform (WO 00/24883; Novozymes A/S).

Kozak, 1981, Nucleic Acids Research 9: 5233-5252, proposed the following“consensus” sequence for initiation of translation in higher eukaryotes:

-   -   Aa Acc aug G        In this sequence, often referred to as a “consensus Kozak”, the        most highly conserved nucleotides are the purines, adenine(A)        and guanine (G), shown in capital letters above; the start-codon        of the gene to be translated is underlined in the above.        Mutational analysis confirmed that these two positions have the        strongest influence on initiation (Kozak, 1987, Molecular Cell        Biology 7: 3438-3445). Kozak also determined that alterations in        the sequence upstream of the consensus Kozak can effect        translation (Kozak, 1986, Proceedings of the National Academy of        Sciences USA 83: 2850-2854).

WO 94/11523 and WO 01/51646 disclose expression vectors comprising afully impaired consensus Kozak or “crippled” consensus Kozak sequence.

SUMMARY OF THE INVENTION

Expression cloning as such in filamentous fungi is presently part of thestandard methodology in the art, however the use of such methods is ofsuch industrial relevance that even minor increments in efficiency,performance or economy is of great interest. Until now expressioncloning in filamentous fungi may have provided an interestingpolypeptide candidate, whereupon the encoding gene would typically havebeen sub-cloned into a more suitable expression vector to achievepolypeptide yields of sufficient quantity to further characterize thepolypeptide of interest, before setting up expensive larger scale trialproductions. A problem to be solved is how to screen a polynucleotidelibrary for a polypeptide with a property of interest in a filamentousfungal host cell in a manner which allows quick and easycharacterization of the subsequent polypeptide.

An aspect of the present invention relates to methods for isolating arecombinant polypeptide of interest, the methods comprising the stepsof:

-   -   a) providing a polynucleotide library derived from an organism        capable of producing one or more polypeptides of interest,        wherein the library was prepared in an expression cloning vector        comprising at least the following elements:

i) a polynucleotide encoding a selectable marker in which thetranslation initiation start site of the marker-encoding sequencecomprises the following sequence: −4  N YNN ATG YNN (SEQ ID NO: 1)

-   -   -   -   wherein “Y” in position −3 is a pyrimidin (Cytidine or                Thymidine/Uridine), “N” is any nucleotide, and the                numerical designations are relative to the first                nucleotide in the start-codon “ATG” (in bold) of the                marker;

        -   ii) a fungal replication initiation sequence, preferably an            automously replicating sequence (ARS), more preferably an            AMA1-sequence or a functional derivative thereof; and

        -   iii) a polynucleotide comprising in sequential order: a            promoter derived from a filementous fungal cell, a            cloning-site into which the library is cloned, and a            transcription terminator;

    -   b) transforming a filamentous fungal host cell with the library;

    -   c) culturing the transformed host cell obtained in (b) under        conditions suitable for expression of the polynucleotide        library; and

    -   d) selecting a transformed host cell which produces the        polypeptide of interest.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of the first aspect of theinvention for isolating a recombinant polypeptide of interest, themethod comprising the steps of:

-   -   a) providing a polynucleotide library derived from an organism        capable of producing one or more polypeptides of interest,        wherein the library was prepared in an expression cloning vector        comprising at least the following elements:

i) a polynucleotide encoding a selectable marker in which thetranslation initiation start site of the marker-encoding sequencecomprises the following sequence: −4  N YNN ATG YNN (SEQ ID NO: 1)

-   -   -   -   wherein “Y” in position −3 is a pyrimidin (Cytidine or                Thymidine/Uridine), “N” is any nucleotide, and the                numerical designations are relative to the first                nucleotide in the start-codon “ATG” (in bold) of the                marker;

        -   ii) a fungal replication initiation sequence, preferably an            automously replicating sequence (ARS), more preferably an            AMA1-sequence or a functional derivative thereof; and

        -   iii) a polynucleotide comprising in sequential order: a            promoter derived from a filementous fungal cell, a            cloning-site into which the library is cloned, and a            transcription terminator;

    -   b) transforming a filamentous fungal host cell with the library;

    -   c) culturing the transformed host cell obtained in (b) under        conditions suitable for expression of the polynucleotide        library; and

    -   d) selecting a transformed host cell which produces the        polypeptide of interest.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide, and under conditions that select for multiple copies of theselectable marker, using methods known in the art. For example, the cellmay be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection).

If the polypeptide of interest is secreted Into the nutrient medium, thepolypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. The polypeptide may be recovered by methodsknown in the art. For example, the polypeptide may be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray-drying, evaporation,or precipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

Crippled Translational Initiator Sequences

The term “translational initiator sequence” is defined herein as the tennucleotides immediately upstream of the initiator or start codon of theopen reading frame of a polypeptide-encoding nucleic acid sequence. Theinitiator codon encodes for the amino acid methionine, the so-called“start” codon. The initiator codon is typically an ATG, but may also beany functional start codon such as GTG. It is well known in the art thaturacil (uridine), U, replaces the deoxynucleotide thymine (thymidine),T, in RNA.

The term “crippled translational initiator sequence” is defined hereinas the ten nucleotides immediately upstream of the initiator codon ofthe open reading frame of a polypeptide-encoding nucleic acid sequence,wherein the initiator sequence comprises a T at the −3 position and a Tat one or more of the −1, −2, and −4 positions.

Accordingly, a preferred embodiment of the invention relates to a methodof the first aspect, wherein the sequence SEQ ID NO:1 comprises aThymidin (Uridin) in the −3 position; even more preferably the thesequence SEQ ID NO:1 further comprises a Thymidin (Uridin) in one moreof the positions −1, −2, and 4.

The term “operably linked” is defined herein as a configuration in whicha control sequence, e.g., a crippled translational initiator sequence,is appropriately placed at a position relative to a coding sequence suchthat the control sequence directs the production of a polypeptideencoded by the coding sequence.

The term “coding sequence” is defined herein as a nucleic acid sequencethat is transcribed into mRNA which is translated into a polypeptidewhen placed under the control of the appropriate control sequences. Theboundaries of the coding sequence are generally determined by the startcodon located at the beginning of the open reading frame of the 5′ endof the mRNA and a stop codon located at the 3′ end of the open readingframe of the mRNA. A coding sequence can include, but is not limited to,genomic DNA, cDNA, semisynthetic, synthetic, and recombinant nucleicacid sequences.

In the methods of the present invention, the crippled translationalinitiator sequence is foreign to the gene encoding a selectable marker.

The crippled translational sequence results in inefficient translationof the gene encoding the selectable marker. When a fungal host cellharbouring an expression vector comprising a polynucleotide encoding apolypeptide of interest physically linked with a second polynucleotidecomprising a crippled translational initiator sequence operably linkedto a gene encoding a selectable marker, is cultured under conditionsthat select for multiple copies of the selectable marker, the copynumber of the polypeptide-encoding polynucleotide cloned into the vectoris also increased.

The term “selectable marker” is defined herein as a gene the product ofwhich provides for biocide or viral resistance, resistance to heavymetals, prototrophy to auxotrophs, and the like, which permits easyselection of transformed cells. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus. Functional derivatives of these selectablemarkers are also of interest in the present invention, in particularthose functional derivatives which have decreased activity or decreasedstability, thereby enabling a selection for a higher copy-number of theexpression vector without increasing the concentration of the selectivesubstance(s).

Accordingly, a preferred embodiment is a method of the first aspect,wherein the selectable marker of step (i) is selected from the group ofmarkers consisting of amdS, argB, bar, hygB, niaD, pyrG, sC, and trpC;preferably the selectable marker of step (i) is pyrG or a functionalderivative thereof, more preferably the selectable marker of step (i) isa functional derivative of pyrG which comprises a substitution of one ormore amino acids, and most preferably the derivative comprises the aminoacid substitution T102N.

The term “copy number” is defined herein as the number of molecules, pergenome, of a gene which is contained in a cell. Methods for determiningthe copy number of a gene are will known in the art and include Southernanalysis, quantitative PCR, or real time PCR.

The fungal host cell preferably contains at least two copies, morepreferably at least ten copies, even more preferably at least onehundred copies, most preferably at least five hundred copies, and evenmost preferably at least one thousand copies of the expression cloningvector.

Polypeptide Encoding Polynucleotides

The polypeptide of interest may be native or heterologous to thefilamentous fungal host cell of interest. The term “heterologouspolypeptide” is defined herein as a polypeptide which is not native tothe fungal cell, a native polypeptide in which modifications have beenmade to alter the native sequence, or a native polypeptide whoseexpression is quantitatively altered as a result of a manipulation ofthe fungal cell by recombinant DNA techniques. The polynucleotideencoding the polypeptide of interest may originate from any organismcapable of producing the polypeptide of interest, includingmulticellular organisms and microorganisms e.g. bacteria and fungi.

A preferred embodiment of the invention relates to methods of the firstaspect, wherein the organism of step (a) capable of producing one ormore polypeptides of interest is a eukaryote, preferably the eukaryoteis a fungus, and most preferably a filamentous fungus.

The term “polypeptide” is not meant herein to refer to a specific lengthof the encoded product and, therefore, encompasses peptides,oligopeptides, and proteins.

Preferably, the polypeptide of interest is an enzyme, an enzyme variant,or a functional derivative thereof, more preferably the enzyme or enzymevariant is an oxidoreductase, transferase, hydrolase, lyase, isomerase,or ligase; and most preferably the enzyme or enzyme variant is anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, a pectinolytic enzyme,peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase.

Preferably, the polypeptide is a hormone or hormone variant or afunctional derivative thereof, a receptor or receptor variant or afunctional derivative thereof, an antibody or antibody variant or afunctional derivative thereof, or a reporter.

In a preferred embodiment, the polypeptide is secreted extracellularly.In a more preferred embodiment, the polypeptide is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred embodiment, the polypeptide is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phospholipase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase.

The nucleic acid sequence encoding a polypeptide of interest may beobtained from any prokaryotic, eukaryotic, or other source. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide isproduced by the source or by a cell in which a gene from the source hasbeen inserted.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide of interest are known in the art and include isolationfrom genomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequence from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR).See, for example, Innis et al, 1990, PCR Protocols: A Guide to Methodsand Application, Academic Press, New York. The cloning procedures mayinvolve excision and isolation of a desired nucleic acid fragmentcomprising the nucleic acid sequence encoding the polypeptide, insertionof the fragment into a vector molecule, and incorporation of therecombinant vector into the mutant fungal cell where multiple copies orclones of the nucleic acid sequence will be replicated. The nucleic acidsequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin,or any combinations thereof.

In the methods of the present invention, the polypeptide may alsoinclude a fused or hybrid polypeptide in which another polypeptide isfused at the N-terminus or the C-terminus of the polypeptide or fragmentthereof. A fused polypeptide is produced by fusing a nucleic acidsequence (or a portion thereof) encoding one polypeptide to a nucleicacid sequence (or a portion thereof) encoding another polypeptide.Techniques for producing fusion polypeptides are known in the art, andinclude, ligating the coding sequences encoding the polypeptides so thatthey are in frame and expression of the fused polypeptide is undercontrol of the same promoter(s) and terminator. The hybrid polypeptidemay comprise a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides wherein one or moremay be heterologous to the mutant fungal cell.

Once a transformed host cell has been selected which produces thepolypeptide of interest according to the methods of the invention, theencoding polynucleotide can be isolated from the selected transformedhost cell, and a further optimized expression system can be designed.

Accordingly, a preferred embodiment relates to methods of the firstaspect, wherein subsequently to step (d) the polynucleotide coding forthe polypeptide of interest is isolated from the selected transformedhost cell of step (d).

Fungal Replication Initiating Sequences

As used herein, the term “fungal replication initiating sequence” isdefined as a nucleic acid sequence which is capable of supportingautonomous replication of an extrachromosomal molecule, e.g., a DNAvector such as a plasmid, in a filamentous fungal host cell, normallywithout structural rearrangement of the DNA-vector or integration intothe host cell genome. The replication initiating sequence may be of anyorigin as long as it is capable of mediating replication intiatingactivity in a fungal cell. For instance the replication initiatingsequence may be a telomer of human origin which confer to the plasmidthe ability to replicate in Aspergillus (Aleksenko and Ivanova, Mol.Gen. Genet. 260 (1998) 159-164). Preferably, the replication initiatingsequence is obtained from a filamentous fungal cell, more preferably astrain of Aspergillus, Fusarium or Alternaria, and even more preferably,a strain of A. nidulans, A. oryzae, A. niger, F. oxysporum or Alternariaaltenata.

A fungal replication initiating sequence may be identified by methodswell-known in the art. For instance, the sequence may be identifiedamong genomic fragments derived from the organism in question as asequence capable of sustaining autonomous replication in yeast,(Ballance and Turner, Gene, 36 (1985), 321-331), an indication of acapability of autonomous replication in filamentous fungal cells. Thereplication initiating activity in fungi of a given sequence may also bedetermined by transforming fungi with contemplated plasmid replicatorsand selecting for colonies having an irregular morphology, indicatingloss of a sectorial plasmid which in turn would lead to lack of growthon selective medium when selecting for a gene found on the plasmid (Gemset al, Gene, 98 (1991) 61-67). AMA1 was isolated in this way. Analternative way to isolate a replication initiating sequence is toisolate natural occurring plasmids (eg as disclosed by Tsuge et al.,Genetics 146 (1997) 111-120 for Alternaria aternata).

Examples of fungal replication initiating sequences include, but are notlimited to, the ANS1 and AMA1 sequences of Aspergillus nidulans, e.g.,as described, respectively, by Cullen, D., et al. (1987, Nucleic AcidsRes. 15: 9163-9175) and Gems, D., et al. (1991, Gene 98: 61-67).

Preferred embodiments relate to methods of the first aspect of theinvention, wherein the fungal replication initiation sequence of step(ii) comprises the nucleic acid sequence set forth in SEQ ID NO:1 or SEQID NO:2 of WO 00/24883, or is a functional derivative thereof,preferably the functional derivative is at least 80% identical to SEQ IDNO:1 or SEQ ID NO: 2 of WO 00/24883.

The term “replication initiating activity” is used herein in itsconventional meaning, i.e. to indicate that the sequence is capable ofsupporting autonomous replication of an extrachromosomal molecule, suchas a plasmid or a DNA vector in a fungal cell.

The term “without structural rearrangement of the plasmid” is usedherein to mean that no part of the plasmid is deleted or inserted intoanother part of the plasmid, nor is any host genomic DNA inserted intothe plasmid. The replication initiating sequence to be used in themethods of the present invention is a nucleotide sequence having atleast 50% identity with the nucleic acid sequence of SEQ ID NO:1 or SEQID NO:2 of WO 00/24883, and is capable of initiating replication in afungal cell; or a subsequence of (a) or (b), wherein the subsequence iscapable of initiating replication in a fungal cell.

In a preferred embodiment, the nucleotide sequence has a degree ofidentity to the nucleic acid sequence shown in SEQ ID NO:1 or SEQ IDNO:2 of WO 00/24883 of at least 50%, more preferably at least 60%, evenmore preferably at least 70%, even more preferably at least 80%, evenmore preferably at least 90%, and most preferably at least 97% identity(hereinafter “homologous polynucleotide”). The homologous polynucleotidealso encompasses a subsequence of SEQ ID NO:1 or SEQ ID NO:2 of WO00/24883 which has replication initiating activity in fungal cells. Forpurposes of the present invention, the degree of identity may besuitably determined by means of computer programs known in the art, suchas GAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch,C. D., (1970), Journal of Molecular Biology, 48, 443-45), using GAP withthe following settings for polynucleotide sequence comparison: GAPcreation penalty of 5.0 and GAP extension penalty of 0.3.

The techniques used to isolate or clone a nucleic acid sequence havingreplication initiating activity are known in the art and includeisolation from genomic DNA or cDNA. The cloning from such DNA can beeffected, e.g., by using methods based on polymerase chain reaction(PCR) to detect cloned DNA fragments with shared structural features.(See, e.g., Innis, et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York.) Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR) may be used.

In preferred embodiment, the replication initiating sequence has thenucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 of WO00/24883, or a respective functional subsequence thereof. For instance,a functional subsequence of SEQ ID NO:1 of WO 00/24883 is a nucleic acidsequence encompassed by SEQ ID NO:1 or SEQ ID NO 2 of WO 00/24883 exceptthat one or more nucleotides from the 5′ and/or 3′ end have beendeleted. Preferably, a subsequence contains at least 100 nucleotides,more preferably at least 1000 nucleotides, and most preferably at least2000 nucleotides. In a more preferred embodiment, a subsequence of SEQID NO:1 of WO 00/24883 contains at least the nucleic acid sequence shownin SEQ ID NO:2 of WO 00/24883.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide comprising a crippled translational initiator sequenceoperably linked to a gene encoding a selectable marker in which the 3′end of the crippled translational initiator sequence is immediatelyupstream of the initiator codon of the gene encoding the selectablemarker. The polynucleotides are operably linked to one or more controlsequences which direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences. Expression will be understood to include any step involved inthe production of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression vector when the nucleic acid construct comprises a secondpolynucleotide encoding a polypeptide of interest and all the controlsequences required for its expression.

An isolated polynucleotide encoding a polypeptide may be furthermanipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleic acid sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleic acid sequencesutilizing recombinant DNA methods are well known in the art.

In the methods of the present invention, the nucleic acid sequences maycomprise one or more native control sequences or one or more of thenative control sequences may be replaced with one or more controlsequences foreign to the nucleic acid sequence for improving expressionof the coding sequence in a host cell.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a polypeptideof interest. Each control sequence may be native or foreign to thenucleic acid sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, crippled translational initiator sequence of thepresent invention, signal peptide sequence, and transcriptionterminator. At a minimum, the control sequences include translationalinitiator sequences, and transcriptional and translational stop signals.The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites or cloning sites facilitatingligation of the control sequences with the coding region of the nucleicacid sequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase, Fusarium oxysporum trypsin-like protease (WO96/00787), as well as the NA2-tpi promoter (a hybrid of the promotersfrom the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase); and mutant, truncated,and hybrid promoters thereof.

A preferred embodiment relates to methods of the first aspect, whereinthe promoter of step (iii) is the promoter from the neutral amylaseencoding gene (NA2) from Aspergillus niger disclosed in WO 89/01969.

The control sequence may be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

A preferred embodiment relates to methods of the first aspect, whereinthe transcription terminator of step (iii) is the terminator from theglucoamylase encoding gene (AMG) from Aspergillus niger (Boel, E.;Hjort, I.; Svensson, B.; Norris, F.; Norris, K. E.; Fiil, N. P.,Glucoamylases G1 and G2 from Aspergillus niger are synthesized from twodifferent but closely related mRNAs. EMBO J. 3: 1097 (1984)).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

A preferred embodiment relates to methods of the first aspect, whereinthe promoter is operably linked, upstream of the cloning-site of step(iii), to the polynucleotide encoding the leader peptide of triosephosphate isomerase (tpiA) from Aspergillus nidulans. (Mcknight G. L.,O'Hara P. J., Parker M. L., “Nucleotide sequence of the triosephosphateisomerase gene from Aspergillus nidulans: Implications for adifferential loss of introns”, Cell 46: 143-147(1986)).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may imply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95133836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a crippled translational initiator sequence operably linkedto a gene encoding a selectable marker in which the 3′ end of thecrippled translational initiator sequence is immediately upstream of theinitiator codon of the gene encoding the selectable marker and a nucleicacid sequence encoding a polypeptide of interest as well as any controlsequences involved in the expression of the sequences.

The various nucleic acid and control sequences described above may bejoined together to produce a recombinant expression vector which mayinclude one or more convenient restriction sites to allow for insertionor substitution of the promoter and/or nucleic acid sequence encodingthe polypeptide at such sites. Alternatively, the nucleic acid sequencemay be expressed by inserting the nucleic acid sequence or a nucleicacid construct comprising the crippled translational initiator sequenceand/or sequence into an appropriate vector for expression. In creatingthe expression vector, the coding sequence is located in the vector sothat the coding sequence is operably linked with a crippledtranslational initiator sequence of the present invention and one ormore appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of a nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.

The vectors of the present invention also contain one or more selectablemarkers which permit easy selection of transformed cells as describedearlier.

For autonomous replication, the vector further comprises an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of origins of replication for use in a yeasthost cell are the 2 micron origin of replication, ARS1, ARS4, thecombination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Theorigin of replication may be one having a mutation which makes itsfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75: 1433).

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The host cell may be any fungal cell useful in the methods of thepresent invention. “Fungi” as used herein includes the phyla Ascomycota,Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworthet al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK) as well as theOomycota (as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a preferred embodiment, the fungal host cell is a filamentous fungalcell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

In a preferred embodiment, the filamentous fungal host cell is a cell ofa species of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, or Trichoderma.

In a more preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioldes, or Fusarium venenatum cell. In another mostpreferred embodiment, the filamentous fungal host cell is a Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

In an even most preferred embodiment, the Fusarium venenatum cell isFusarium venenatum A3/5, which was originally deposited as Fusariumgraminearum ATCC 20334 and recently reclassifled as Fusarium venenatumby Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; aswell as taxonomic equivalents of Fusarium venenatum regardless of thespecies name by which they are currently known. In another preferredembodiment, the Fusarium venenatum cell is a morphological mutant ofFusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosedin WO 97/26330.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Example 1

In order to improve expression of a gene of interest on an expressionplasmid, it may be desirable to reduce the expression of the selectiongene, exemplified here by the pyrG gene. By cultivating a host cellharbouring an expression plasmid comprising a selection gene, that hasreduced expression, under normal selective pressure results in aselection for a host cell which has an increased plasmid copy number,thus achieving the total expression level of the selection genenecessary for survival. The higher plasmid copy-number however alsoresults in an increased expression of the gene of interest.

One way of decreasing the expression level of the selection gene is tolower the mRNA level by either using a poorly transcribed promoter ordecreasing the functional halflife of the mRNA. Another way is to reducetranslation efficiency of the mRNA. One way to do this is to mutate theKozak-region. This is a region just upstream of the initiation codon(ATG), which is important for the initiation of translation.

Plasmid pENI2155 comprises a bad kozak region upstream of the pyrG gene,and is constructed as follows:

Using plasmid pENI1861 (the construction of which is described below) astemplate, and PWO polymerase (conditions as recommended bymanufacturer); two PCR-reactions were made using primer 141200j1 and270999J9 in the one PCR-reaction and primers 141200J2 and 290999J8 inanother PCR-reaction: 141200J1 (SEQ ID NO: 2): 5′atcggttttatgtcttccaagtcgcaattg 141200J2 (SEQ ID NO: 3): 5′cttggaagacataaaaccgatggaggggtagcg 270999J8 (SEQ ID NO: 4): 5′tctgtgaggcctatggatctcagaac 270999J9 (SEQ ID NO: 5): 5′gatgctgcatgcacaactgcacctcag

The PCR fragments were purified from a 1% agarose gel using QIAGEN™ spincolumns. A second PCR-reaction was run using the two fragments astemplate along with the primers 270999J8 and 270999J9. The PCR-fragmentfrom this reaction was purified from a 1% agarose gel as described; thefragment and the vector pENI1849 (containing a lipase gene as expressionreporter) were cut with the restricton enzymes StuI and SphI, theresulting fragments were purified from a 1% agarose gel as describedpreviously.

The purified fragments were ligated and transformed into the E. colistrain DH10B. Plasmid DNA from one of the transformants was isolated andsequenced to confirm the introduction of a mutated Kozak region:ggttttatg (rather than the wildtype: gccaacatg). This Plasmid wasdenoted: pENI2155.

Aspergillus cells were transformed with plasmid pENi1849 (controlwildtype plasmid), and pENi2155 (mutated Kozak region upstream of thepyrg gene). Approximately 1 microgram of pENI1849 and pENi2155 weretransformed into A. oryzae Jal355 (JaL355 is a derivative of A. oryzaeA1560 wherein the pyrG gene has been inactivated, as described in WO98/01470; transformation protocol as described in WO 00/24883). Thetransformants were incubated for 4 days at 37° C. 24 transformants fromthe pENi2155 transformation and 12 transformants from pENI1849 wereinoculated in a 96 well microtiter plate containing 1*Vogel medium and2% maltose (Methods in Enzymology, vol. 17, p. 84). After 4 days growthat 34° C., the culture broth was assayed for lipase activity usingpnp-valerate as a lipase substrate.

A 10 microliter aliquot of media from each well was added to amicrotiter well containing 200 microliter of a lipase substrate of0.018% p-nitrophenylvalerate, 0.1% Triton X™-100, 10 mM CaCl₂, 50 mMTris pH 7.5. Lipase activity was assayed spectrophotometrically at15-second intervals over a five minute period, using a kineticmicroplate reader (Molecular Device Corp., Sunnyvale Calif.), using astandard enzymology protocol (e.g., Enzyme Kinetics, Paul C. Engel, ed.,1981, Chapman and Hall Ltd.). Briefly, product formation is measuredduring the initial rate of substrate turnover and is defined as theslope of the curve calculated from the absorbance at 405 nm every 15seconds for 5 minutes. The arbitrary lipase activity units werenormalized against the transformant showing the highest lipase activity.For each group of thirty transformants an average value and the standarddeviations were calculated. Given in arbitrary units the average lipaseactivity and relative standard deviation was:

-   1849 Transformant: 65±14-   2155 Transformant: 120±22

Clearly there is nearly a doubling of lipase expression in the 2155transformant, wherein the mutated Kozak region was introduced in frontof the selection gene pyrG.

Plasmid pENI1861 was made in order to have the state of the artAspergillus promoter in the expression plasmid, as well as a number ofunique restriction sites for cloning. A PCR fragment (Approx. 620 bp)was made using plasmid pMT2188 (the construction of pMT2188 is describedbelow) as template and the following primers: 051199J1 (SEQ ID NO: 6):5′ cctctagatctcgagctcggtcaccggtggcctccgcggccgctggatccccagttgtg 1298TAKA(SEQ ID NO: 7): 5′ gcaagcgcgcgcaatacatggtgttttgatcat

The fragment was cut with BssHII and BglII, and cloned into pENI1849which was also cut with BssHII and Bgl II. The cloning was verified bysequencing.

Plasmid pENI1849 was made in order to truncate the pyrG gene to theessential sequences for pyrG expression, in order to decrease the sizeof the plasmid, thus improving transformation frequency. A PCR fragment(Approx. 1800 bp) was made using pENI1299 (described in WO 00/24883 FIG.2 and Example 1) as template and the following primers: 270999J8 (SEQ IDNO:3), and 270999J9 (SEQ ID NO:4).

The PCR-fragment was cut with the restriction enzymes StuI and SphI, andcloned into pENI1298 (described in WO 00/24883 FIG. 1 and Example 1),also cut with StuI and SphI; the cloning was verified by sequencing.

Plasmid pMT2188 was based on the Aspergillus expression plasmid pCaHj483 (described in WO 98/00529) which consists of an expression cassettebased on the Aspergillus niger neutral amylase 11 promoter fused to theAspergillus nidulans triose phosphate isomerase non translated leadersequence (Pna2/tpi) and the A. niger amyloglycosidase terminater (Tamg).Also present on the pCaHj483 is the Aspergillus selective marker amdSfrom A. nidulans enabling growth on acetamide as sole nitrogen source.These elements are cloned into the E. coli vector pUC19 (New EnglandBiolabs). The ampicillin resistance marker enabling selection in E. coliof pUC19 was replaced with the URA3 marker of Saccharomyces cerevisiaethat can complement a pyrF mutation in E. coli, the replacement was donein the following way:

The pUC19 origin of replication was PCR amplified from pCaHj483 with theprimers: 142779 (SEQ ID NO: 8): 5′ ttgaattgaaaatagattgatttaaaacttc142780 (SEQ ID NO: 9): 5′ ttgcatgcgtaatcatggtcatagc

Primer 142780 introduces a BbuI site in the PCR fragment. The Expand™PCR system (Roche Molecular Biochemicals, Basel, Switserland) was usedfor the amplification following the manufacturers instructions for thisand the subsequent PCR amplifications.

The URA3 gene was amplified from the general S. cerevisiae cloningvector pYES2 (Invitrogen corporation, Carlsbad, Calif., USA) using theprimers: 140288 (SEQ ID NO: 10): 5′ ttgaattcatgggtaataactgatat 142778(SEQ ID NO: 11): 5′ aaatcaatctattttcaattcaattcatcatt

Primer 140288 introduces an EcoRI site in the PCR fragment. The two PCRfragments were fused by mixing them and amplifying using the primers142780 and 140288 in the splicing by overlap method (Horton et al (1989)Gene, 77, 61-68).

The resulting fragment was digested with EcoRI and BbuI and ligated tothe largest fragment of pCaHj 483 digested with the same enzymes. Theligation mixture was used to transform the pyrF E. coli strain DB6507(ATCC 35673) made competent by the method of Mandel and Higa (Mandel, M.and A. Higa (1970) J. Mol. Biol. 45, 154). Transformants were selectedon solid M9 medium (Sambrook et. al (1989) Molecular cloning, alaboratory manual, 2. edition, Cold Spring Harbor Laboratory Press)supplemented with 1 g/l casaminoacids, 500 microgram/l thiamine and 10mg/l kanamycin. A plasmid from a selected transformant was termedpCaHj527. The Pna2/tpi promoter present on pCaHj527 was subjected tosite directed mutagenises by a simple PCR approach. Nucleotide 134-144was altered from GTACTAAAACC to CCGTTAAATTT using the mutagenic primer141223. Nucleotide 423-436 was altered from ATGCAATTTAAACT toCGGCAATTTAACGG using the mutagenic primer 141222. The resulting plasmidwas termed pMT2188. Primer 141223 (SEQ ID NO: 12): 5′ggatgctgttgactccggaaatttaacggtttggtcttgcatccc Primer 141222 (SEQ ID NO:13): 5′ ggtattgtcctgcagacggcaatttaacggcttctgcgaatcgc

Example 2

In order to improve expression of a gene of interest from a plasmid, itmay be desirable to reduce the stability and/or the activity of theprotein encoded by the selection gene (for instance the pyrG gene) asalready mentioned in Example 1.

One way of decreasing the stability of the protein encoded by theselection gene is to add a “degron” motif to the protein (Dohmen R. J.,Wu P., Varshavsky A., (1994) Science vol 263 p. 1273-1276). Another wayis to identify structurally important conserved amino acid residues,based on alignment to homologous proteins or based on a model-structureof the protein (if available). These amino acids may then be mutated todecrease the stability and/or the activity of the enzyme.

A protein alignment was made with the protein sequence:swissprot_dcop_aspng (the OMP decarboxylase encoded by the pyrG gene onplasmid pENI2155) to the following database entries:Swissprot_dcop_aspor, geneseqp_r05224, geneseqp_y99702,tremblnew_aag34761, swissprot_dcop_phybl, remternbl_aab01165,remtembl_aab16845, and sptrembl_q9uvz5.

The alignment was done using the program ClustalW (Thompson, J. D.,Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, positions-specific gap penalties and weight matrix choice.Nucleic Acids Research, 22: 4673-4680).

Based on these alignments and the structure of the related Bacillussubtilis OMP decarboxylase (Appleby t., Kinsland C., Begley T. P.,Ealick S. E. (2000), Proc. Natl. Acad. Sci. USA, vol 97 p. 2005-2010)the following conserved residues were identified as potentiallystructurally important, and as such suitable targets for mutation: P50,F91, F96, N101, T102, G128, G222, D223, G239. A number of mutagenicprimers were constructed, and were phosphorylated using T4polynucleotide kinase (New England Biolabs). P50 - 260301j1 (SEQ ID NO:14): 5′ acaggactcggtncgtacattgccgtg F91 - 260301j2 (SEQ ID NO: 15): 5′aatttcctcatctncgaagatcgcaag F96 - 260301j3 (SEQ ID NO: 16): 5′gaagatcgcaagtncatcgatatcgga N101, T102 - 260301j4 (SEQ ID NO: 17): 5′atcgatatcgganacancgtccaaaagcag G128 - 260301j5 (SEQ ID NO: 18): 5′agtattctgcccgntgagggtatcgtc G222, D223 - 260301j6 (SEQ ID NO: 19): 5′ctctcctcgaaggntnacaagctgggacag G239 - 230301j7 (SEQ ID NO: 20): 5′gctgttggacgcgntgccgactttatt

Seven individual PCR/ligation reactions were performed (as described bySawano A., Miyawaki A. (2000) Nucleic Acid Research vol 28 e78) usingpENI2155 as template, and 1 microliter DNA from each of the sevenlibraries was transformed into the E. coli strain DH10B. Approximately1000 E. coli clones were obtained from each library. DNA preparation wasmade from each library and the DNA was pooled together (named pBIB16).

The Aspergillus strain MT2425 (a pyrG minus strain, which gives smalltransformant-clones, when grown on the selection plates) was transformedwith 1 microgram of the pBIB16 DNA and 10 microgram herring sperm DNA(carrier DNA) pr. 100 microliter protoplast using standard procedures.

The transformed protoplast were spread on selection plates (2% maltose(inducing small morphology and lipase expression), 10 mM NaNO₃, 1.2 Msorbitol, 2% bacto agar, and standard salt solution.

After 5 days of growth, an overlay (containing 0.004% brilliant green,2.5% olive oil, 1% agar, 50 mM TRIS pH 7.5 treated with a mixer for 1min. (Ultrathorax™ Type T25B, IKA Labortechnic, Germany)) was pouredonto the Aspergillus transformant clones. The plates where incubatedover night at room temperature.

Twenty of the clones having highest activity towards olive oil wereinoculated in to 200 microliter YPM in a 96 well microliter plate. After4 days of growth at 34° C., the culture broths were assayed for lipaseactivity using pnp-valerate as described above.

The 6 transformants giving the highest activity in the lipase assay wereinoculated in 5 ml YPM. DNA was isolated and transformed into the E.coli strain DH10B, thus rescuing the plasmid (as also described in WO00/24883). Two pyrG variants were identified:

-   -   1) F96S; the plasmid was denoted pENI2343, and    -   2) T102N; the plasmid was denoted pENI2344.

Approx. 2 microgram of each of the plasmids pENI2155, pENI2343 andpENI2344 were transformed into an Aspergillus oryzae pyrG-minus mutantdenoted Jal355, and an Aspergillus niger pyrG-minus mutant denotedMbin115, using standard procedures.

The transformed protoplasts were spread on selection plates (2% maltose10 mM NaNO₃, 1.2 M sorbitol, 2% bacto agar, salt solution. After 4 daysof growth, very poor sporulatlon was seen for the pENI2343 Jal355transformants, and no transformants were seen for MBIN115 transformedwith pENI2343.

6 independent transformants of each plasmid transformation wereinoculated into 200 microliter 1*vogel, 2% maltose in a 96-wellmicroliter plate. After 4 days growth at 34° C., the culture broths wereassayed for lipase activity. The results are given in the table below asrelative lipase units with relative standard deviation, and are averagesof the activity of the independent clones. Jal355 Mbin115 pENI2155 (wt)48 ± 8%  7 ± 14% pENI2343 (F96S) 49 ± 15% No growth pENI2344 (T102N) 71± 13% 80 ± 11%

The expression of lipase from the pENI2343 transformants was very highcompared to the fungal biomass in the wells, which was very poor (lessthan 1/10 of the other transformants). An approx. 1.5-fold increase inlipase expression level is seen for the Jal355 transformants, and anapprox. 11-fold increase is seen in the Mbin115 transformants, whencomparing the pENI2155 transformants with the pENI2344 transformants.

Thus the pyrG T102N mutation leads to an increase in lipase expression,likely due to an increased plasmid copy number, which is selected forbecause of the unstable, less active OMP decarboxylase encoded by theselection gene pyrG.

Example 3

In order to evaluate plasmid stability, a screen was set up to evaluatethe percentage of spores containing a stably episomaly replicatedplasmid (comprising a pyrG selection gene).

Two DNA libraries were constructed, the first library was cloned into aplasmid comprising the wildtype pyrG gene as selection gene, whereas thesecond library was cloned into a plasmid comprising a mutated pyrg genewhich comprised a mutated Kozak region as described in Example 1 and aT102N mutation as described in Example 2.

A spore suspension was made from each library and plated on to growthplates (2% maltose 10 mM NaNO₃, 1.2 M sorbitol, 2% bacto agar, salts,with or without 20 mM uridine). The plates were grown for 3 days at 37°C. Results are shown in the table below. Selection gene −uridine+uridine % viable spores Wildtype pyrG 11 83 13 Mutant (Kozak/T102N)pyrG 36 63 57

Evidently a much larger fraction of the spores contain a plasmid, whenusing the mutated (Kozak/T102N) pyrG gene.

1-19. (canceled)
 20. A method for isolating a recombinant polypeptide ofinterest, the method comprising the steps of: a) providing apolynucleotide library derived from an organism capable of producing oneor more polypeptides of interest, wherein the library was prepared in anexpression cloning vector comprising at least the following elements: i)a polynucleotide encoding a selectable marker in which the translationinitiation start site of the marker-encoding sequence comprises thefollowing sequence: −4 N YNN ATG YNN (SEQ ID NO: 1) wherein “Y” inposition −3 is a pyrimidin (Cytidine or Thymidine/Uridine) and “N” isany nucleotide; ii) a fungal replication initiation sequence, preferablyan autonomously replicating sequence (ARS), more preferably anAMA1-sequence or a functional derivative thereof; and iii) apolynucleotide comprising in sequential order: a promoter derived from afilamentous fungal cell, a cloning-site into which the library iscloned, and a transcription terminator; b) transforming a filamentousfungal host cell with the library; c) culturing the transformed hostcell obtained in (b) under conditions suitable for expression of thepolynucleotide library; and d) selecting a transformed host cell whichproduces the polypeptide of interest.
 21. The method of claim 20,wherein the organism of step (a) is capable of producing one or morepolypeptides of interest is a eukaryote.
 22. The method of claim 21,wherein the eukaryote is a fungus.
 23. The method of claim 20, whereinthe sequence (SEQ ID NO: 1) comprises a Thymidin (Uridin) in the −3position.
 24. The method of claim 23, wherein the sequence (SEQ IDNO: 1) further comprises a Thymidin (Uridin) in one or more of thepositions −1, −2, and −4.
 25. The method of claim 20, wherein theselectable marker of step (i) is selected from the group of markersconsisting of amdS, argB, bar, hygB, niaD, pyrG, sC, and trpC.
 26. Themethod of claim 25, wherein the selectable marker of step (i) is pyrG ora functional derivative thereof.
 27. The method of claim 26, wherein theselectable marker of step (i) is a functional derivative of pyrG whichcomprises a substitution of one or more amino acids, preferably thederivative comprises the amino acid substitution T102N.
 28. The methodof claim 20, wherein the fungal replication initiation sequence of step(ii) comprises the nucleic acid sequence set forth in SEQ ID NO: 1 orSEQ ID NO: 2 of WO 00/24883, or is a functional derivative thereof,preferably the functional derivative is at least 80% identical to SEQ IDNO: 1 or SEQ ID NO: 2 of WO 00/24883.
 29. The method of claim 20,wherein the promoter of step (iii) is the promoter from the neutralamylase encoding gene (NA2) from Aspergillus niger disclosed in WO89/01969.
 30. The method of claim 29, wherein the promoter is operablylinked, upstream of the cloning-site of step (iii), to thepolynucleotide encoding the leader peptide of triose phosphate isomerase(tpiA) from Aspergillus nidulans.
 31. The method of claim 20, whereinthe transcription terminator of step (iii) is the terminator from theglucoamylase encoding gene (AMG) from Aspergillus niger.
 32. The methodof claim 20, wherein the filamentous fungal host cell is of the genusAcremonium, Aspergillus, Coprinus, Fusarium, Humicola, Mucor,Myceliopthora, Neurospora, Penicillium, Thielavia, Tolypocladium orTrichoderma.
 33. The method of claim 32, wherein the cell is of thespecies Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans,Coprinus cinereus, Fusarium oxysporum, or Trichoderma reesei.
 34. Themethod of claim 20, wherein the polypeptide of interest is an enzyme.35. The method of claim 34, wherein the enzyme is an enzyme variant. 36.The method of claim 34, wherein the enzyme or enzyme variant is anoxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. 37.The method of claim 34, wherein the enzyme or enzyme variant is anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, a pectinolytic enzyme,peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase.
 38. The method of claim 20,further comprising isolating the polynucleotide coding for thepolypeptide of interest from the selected transformed host cell of step(d).